Alloreactivity, the ability of a T cell to recognize foreign MHC molecules, to which the T cell had never been exposed, occurs in 1-10% of unprimed T cells, and is observed in vivo as graft rejection, or graft versus host disease, and in vitro as a mixed lymphocyte reaction (MLR). The phenomenon of alloreactivity has been known for a long time;however, the precise molecular basis for it is yet to be fully defined. In these proposed studies, we want to test the hypothesis that peptide recognition by allogeneic T cells is represented by a continuum of specificity, which overlaps with conventional syngeneic recognition, but contains a nonoverlapping area of degenerate recognition. Previous studies from our laboratory on the peptide specificity of two class II alloresponses have revealed one T cell, 2.102, where the recognition is degenerate compared to the syngeneic response. Conversely, for a second allo TCR, KRN, the allogeneic peptide recognition is as specific as the syngeneic recognition. For a third T cell, 3.L2, we have generated a high affinity variant using a yeast display system. This high affinity TCR, M15, exhibits peptide degeneracy compared to 3.L2. Importantly, we have solved a high resolution (2.3A) crystal structure of M15 bound its Hb/I-Ek ligand. The 3.L2 and Ml5 pair represent specific and degenerate peptide recognition respectively, and can therefore be used to model a specific syngeneic and degenerate alloresponse. The crystal structure provides a strong structural basis to explore the peptide recognition and degeneracy. In these proposed studies we want to test whether alloreactivity is biased towards degenerate recognition. We are proposing a series of novel, integrated, and comprehensive studies in which we will examine peptide specificity in multiple ways. In Aim I, we propose to identify and characterize the naturally presented self-peptides in a series of unprimed alloreactive T cells. In Aim II, we will perform a novel gain of function approach using a yeast display system in which we will create TCRs which have acquired I-EP alloreactivity. From these studies, we can ascertain what changes in a TCR are required to make it alloreactive and what changes in peptide specificity have occurred. In Aim III, we propose to study the peptide specificity/degeneracy of the 3.L2 and M15 TCRs structurally and biophysically. Overall, these studies will provide key insights into the molecular basis for the degenerate vs. specific peptide recognition and will advance our understanding of alloreactivity.